Standard- and large-sized eggs of Trichuris trichiura in the feces of schoolchildren in the Yangon Region, Myanmar: Morphological and molecular analyses.

Standard- and large-sized eggs of Trichuris trichiura were found in the feces of schoolchildren in Yangon, Myanmar during epidemiological surveys and mass deworming with albendazole in 2017-2019. The standard-sized eggs were identified as those of T. trichiura, but it was necessary to exclude the possibility of the large-sized eggs belonging to Trichuris vulpis, a dog whipworm. We conducted morphological and molecular studies to determine the species of the 2 types of Trichuris eggs. Individual eggs of both sizes were isolated from Kato-Katz fecal smears (n=20) and mechanically destroyed using a 23G injection needle. Nuclear DNA was extracted, and the 18S rRNA region was sequenced in 15 standard-sized eggs and 15 large-sized eggs. The average size of standard-sized eggs (T. trichiura) was 55.2×26.1 µm (range: 51.7-57.6×21.3-28.0 µm; n=97), whereas the size of large-sized eggs was 69.3×32.0 µm (range: 65.1-76.4×30.1-34.5 µm; n=20), slightly smaller than the known size of T. vulpis. Regarding standard-sized eggs, the 18S rRNA nucleotide sequences exhibited 100% homology with T. trichiura deposited in GenBank and 88.6-90.5% homology with T. vulpis. Regarding large-sized eggs, the nucleotide sequences showed 99.8-100% homology with T. trichiura in GenBank and 89.6-90.7% homology with T. vulpis. Both standard- and large-sized eggs of Trichuris spp. found in Myanmar schoolchildren during 2017-2019 were morphologically and molecularly confirmed to belong to T. trichiura. The conversion of eggs from smaller to large sizes might be due to anthelmintic treatments with albendazole.

Exosomal miRNA-21 from Toxoplasma gondii-infected microglial cells induces the growth of U87 glioma cells by inhibiting tumor suppressor genes.

Toxoplasma gondii is an intracellular protozoan parasite that can modulate the microenvironment of infected hosts and is known to be associated with the incidence of brain tumor growth. In this study, we suggested that the exosomal microRNA-21 derived from Toxoplasma infection would contribute to the growth of brain tumors. Exosomes of BV2 microglial cells infected with Toxoplasma were characterized and confirmed internalization to U87 glioma cells. Exosomal miRNA expression profiles were analyzed using microRNA array and miR-21A-5p associated with Toxoplasma and tumor sorted. We also examined the mRNA level of tumor-associated genes in U87 glioma cells by changing the level of miR-21 within exosomes and the effects of exosomes on the proliferation of human U87 glioma cells. Expression of miRNA-21 was increased and anti-tumorigenic genes (FoxO1, PTEN, and PDCD4) were decreased in exosomes within T. gondii-infected U87 glioma cells. Toxoplasma-infected BV2-derived exosomes induced proliferation of U87 glioma cells. The exosomes induced the growth of U87 cells in a mouse tumor model. We suggest that the increased exosomal miR-21 from Toxoplasma-infected BV2 microglial cells may play an important role as a cell growth promotor of U87 glioma cells through a down-regulation of anti-tumorigenic genes.

Molecular Diagnosis of Pseudoterranova decipiens Sensu Stricto Infections, South Korea, 2002‒2020.

Human Pseudoterranova decipiens larval infections were diagnosed by molecular analysis of mitochondrial cox1 and nd1 genes in 12 health check-up patients in South Korea during 2002-2020. Based on high genetic identity (99.3%-100% for cox1 and 96.7%-98.0% for nd1), we identified all 12 larvae as P. decipiens sensu stricto.

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams, Ruditapes philippinarum, from Aphae-do (Island), Shinan-gun, Korea.

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphaedo (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307-570 (av. 395) µm long and 101-213 (av. 157) µm wide. Most of the daughter sporocysts contained 15-20 furcocercous cercariae each. The cercariae measured 112-146 (av. 134) µm in total length and 35-46 (av. 40) µm in width, with 69-92 (av. 85) µm long body and 39-54 (av. 49) µm long tail. The metacercariae were 210-250 (av. 231) µm in length and 170-195 (av. 185) µm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1-5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8-99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.

Failure of Repeated MDA with Albendazole for Trichuriasis Control in Schoolchildren of the Yangon Region, Myanmar.

Soil-transmitted helminth (STH) infections are still a considerable challenge in Myanmar. We undertook a control program for STH infections (especially Trichuris trichiura) among schoolchildren in Myanmar using mass drug administration (MDA) and health education. Around 1,700 schoolchildren from 15 primary schools in 3 suburban districts (Shwe Pyi Thar, Twantay, and Kyauktan) of the Yangon Region were subjected in this study during 2017-2019. All of the schoolchildren in each school were orally administered albendazole (400 mg in a single dose) 2, 3, and 4 times a year in 2017, 2018, and 2019, respectively. The results revealed that the egg positive rate of any intestinal helminths (including STH) was reduced from 37.6% (649/1,724) in 2017 to 22.8% (352/1,542) in 2019. The egg positive rate of Ascaris lumbricoides was decreased remarkably from 23.3% (402/1,724) in 2017 to 3.6% (56/1,542) in 2019. However, that of T. trichiura was only slightly reduced from 26.9% (464/1,724) in 2017 to 20.2% (312/1,542) in 2019. The intensity of infection with A. lumbricoides and T. trichiura was both more or less reduced, and the proportion of light infection cases with A. lumbricoides and T. trichiura increased from 35.6% in 2017 to 64.3% in 2019 and from 70.3% in 2017 to 81.7% in 2019, respectively. The results indicated that repeated MDAs (2-4 times a year for 3 years) using albendazole on schoolchildren in Myanmar failed to control T. trichiura infection. For a successful control of trichuriasis in Myanmar, new MDA strategies, using a modified albendazole regimen (multiple daily doses for 2 or 3 days) or an alternative anthelmintic drug, such as oxantel pamoate, is strongly recommended.

Neuroprotective Effect of Chronic Intracranial Toxoplasma gondii Infection in a Mouse Cerebral Ischemia Model.

Toxoplasma gondii is an obligate intracellular protozoan parasite that can invade various organs in the host body, including the central nervous system. Chronic intracranial T. gondii is known to be associated with neuroprotection against neurodegenerative diseases through interaction with host brain cells in various ways. The present study investigated the neuroprotective effects of chronic T. gondii infection in mice with cerebral ischemia experimentally produced by middle cerebral artery occlusion (MCAO) surgery. The neurobehavioral effects of cerebral ischemia were assessed by measurement of Garcia score and Rotarod behavior tests. The volume of brain ischemia was measured by triphenyltetrazolium chloride staining. The expression levels of related genes and proteins were determined. After cerebral ischemia, corrected infarction volume was significantly reduced in T. gondii infected mice, and their neurobehavioral function was significantly better than that of the uninfection control group. Chronic T. gondii infection induced the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in the brain before MCAO. T. gondii infection also increased the expression of vascular endothelial growth factor after the cerebral ischemia. It is suggested that chronic intracerebral infection of T. gondii may be a potential preconditioning strategy to reduce neural deficits associated with cerebral ischemia and induce brain ischemic tolerance through the regulation of HIF-1α expression.

Immunogenicity of Exosomes from Dendritic Cells Stimulated with Toxoplasma gondii Lysates in Ocularly Immunized Mice.

Immunogenicity of dendritic cell-derived exosomes stimulated with Toxoplasma gondii lysates (TLA exo), mixed with cholera toxin as an adjuvant, was investigated in mice immunized via 2 mucosal routes (ocular vs intranasal). BALB/c mice were injected 3 times with TLA exo vaccine at 2 week interval, and the levels of IgG in serum and IgA in tear, saliva, feces, and vaginal wash were measured. To observe the expression of T. gondii-specific B1 gene, mice infected with ME49 T. gondii cysts were immunized with TLA exo or PBS exo (not stimulated with TLA), and their brain tissues were examined. The mice vaccinated via intranasal route elicited significantly higher humoral and mucosal immune responses compared with mice treated with PBS alone. Also, mice immunized via ocular route (by eyedrop) induced significantly higher T. gondii-specific IgG in serum and IgA in tear and feces in comparison with PBS controls. B1 gene expression was significantly lower in TLA exo vaccinated mice than in PBS or PBS exo vaccinated mice. These results demonstrated that ocular immunization of mice with TLA exo vaccine has the potential to stimulate systemic or local antibody responses. This study also highlighted an advantage of an eyedrop vaccine as an alternative for T. gondii intranasal vaccines.

Rare Case of Enteric Ancylostoma caninum Hookworm Infection, South Korea.

A 60-year-old man from South Korea underwent a colonoscopy. A juvenile female worm showing 3 pairs of teeth in the buccal cavity was recovered from the descending colon. Partial sequencing of the internal transcribed spacer region showed 100% identity with Ancylostoma caninum, the dog hookworm.

Neurocysticercosis: Clinical Characteristics and Changes from 26 Years of Experience in an University Hospital in Korea.

The prevalence of human taeniasis has decreased in Korea. The stool egg positive proportion decreased from 1.9% in 1971 to 0% in 2004 in nationwide surveys. The neurocysticercosis (NCC) is also presumed to decrease. However, detailed information regarding the recent status of NCC in Korea is lacking. We retrospectively reviewed NCC cases from 1990 to 2016 at Asan Medical Center, a 2700-bed tertiary referral hospital in Korea. We identified patients based on clinical symptoms, brain imaging, pathology and serological assay. The cases were classified as parenchymal, extraparenchymal, and mixed NCC. Eighty-one patients were included in the analysis. The mean age was 54.5 years, and 79.0% were male. The number of NCC cases was highest from 1995 to 1999, and continuously decreased thereafter. Forty (49.4%) patients had parenchymal NCC, while 25 (30.9%) patients had extraparenchymal NCC, and 16 (19.8%) patients had mixed NCC. The seizure and headache were most common symptom of parenchymal NCC and extraparenchymal NCC respectively. Hydrocephalus was more common in extraparenchymal NCC, and patients with extraparenchymal NCC were more likely to require a ventriculoperitoneal shunt. Cases of NCC are decreasing accordingly with human taeniasis and lesion location was the most important determinant of clinical presentation and outcome of NCC in Korea.

Molecular Identification of Anisakis Larvae Extracted by Gastrointestinal Endoscopy from Health Check-up Patients in Korea.

Anisakiasis is a zoonotic disease induced by anisakid nematodes, and endoscopic inspection is used for a diagnosis or remedy for it. Anisakis simplex, Anisakis physeteris, and Pseudoterranova decipiens had been reported to be the major species causing human infections, particularly, in Japan. However, in Korea, recent studies strongly suggested that Anisakis pegreffii is the major species of human infections. To support this suggestion, we collected anisakid larvae (n=20) from 20 human patients who were undergone gastrointestinal endoscopy at a health check-up center in Korea, and molecular identification was performed on the larvae using PCR-RFLP analysis and gene sequencing of rDNA ITS regions and mtDNA cox2. In addition, anisakid larvae (n=53) collected from the sea eel (Astroconger myriaster) were also examined for comparison with those extracted from humans. The results showed that all human samples (100%) were identified as A. pegreffii, whereas 90.7% of the samples from the sea eel were A. pegreffii with the remaining 9.3% being Hysterothylacium aduncum. Our study confirmed that A. pegreffii is the predominant species causing human anisakiasis in Korea, and this seems to be due to the predominance of this larval type in the fish (sea eels) popularly consumed by the Korean people. The possibility of human infection with H. aduncum in Korea is also suggested.

Survey of Gymnophalloides seoi Metacercariae in Natural and Cultured Oysters from Several Western Coastal Areas, Korea.

Gymnophalloides seoi (Digenea: Gymnophallidae) is a human intestinal trematode contracted by eating raw oysters (Crassostrea gigas) in the Republic of Korea (=Korea). It has been known to be highly endemic in Aphae Island, Shinan-gun, Jeollanam-do (Province). However, recent epidemiological status of G. seoi has not been reported since the 1990s. In this study, we investigated the prevalence of G. seoi metacercariae in natural and cultured oysters collected from 3 islands and 2 coastal areas in western parts of Korea. The oysters were examined using the artificial digestion method followed by stereomicroscopy. The overall positive rate of G. seoi metacercariae in natural oysters was 66.0% (99/150), and the oysters collected from Yubu Island showed the highest infection rate (74.0%). However, the metacercarial density per oyster was relatively low (1.5-2.4 per oyster). By contrast, no metacercaria was found in cultured oysters purchased from 2 coastal areas in Chungcheongnam-do. Thus, we could confirm that natural oysters produced from 3 western coastal islands are infected with G. seoi metacercariae, whereas cultured oysters purchased from 2 coastal areas were free from infection.

Worm expulsion of Gymnophalloides seoi from C57BL/6 mice: role of metacercarial exosomes in upregulating TLR2 and MUC2 expression in intestinal tissues.

Gymnophalloides seoi worms were rapidly expelled from C57BL/6 mice within days 3-6 post-infection probably due to operation of mucosal innate immunity. To understand better the mucosal immunity related to worm expulsion from the host, we isolated exosomes of G. seoi metacercariae and investigated their role in induction of mRNA and protein expression of several Toll-like receptors and mucin-related factors in vitro. G. seoi-secreted exosomes were collected using differential ultracentrifugation, and cellular internalization of the exosomes into HT-29 intestinal epithelial cells was visualized by confocal microscopy. The expression of TLR2 and MUC2 in HT-29 cells was up-regulated in stimulation with the exosomes. We suggest that G. seoi-secreted exosomes offer a new point of view in the mechanism of worm expulsion from the host through enhancement of TLR2 and MUC2 expression.

Molecular Diagnosis of Taenia saginata Tapeworm Infection in 2 Schoolchildren, Myanmar.

Taenia saginata is the most common human tapeworm worldwide but has been unknown in Myanmar. In 2017, fecal examination in Yangon, Myanmar, revealed eggs of Taenia species in 2 children from a monastic school. Several proglottids expelled after medication with praziquantel were morphologically and molecularly confirmed to be T. saginata tapeworms.

Seroprevalence and Risk Factors of Toxoplasma gondii Infection among Cat Sitters in Korea.

The seroprevalence of human toxoplasmosis has been increasing in Korea, and it is controversial whether cats are an important infection source or not. This study was performed to evaluate the seroprevalence of Toxoplasma gondii infection in a high risk group (cat sitters) and to determine the possible importance of cats as an infection source in Korea. Risk factors, including the age, sex, and diet of cat sitters, their contact experience and contact frequency with stray cats, and origin, number, and outdoor activity of their pet cats, were analyzed using structured questionnaires. A total of 673 serum samples from people who have frequent contact with cats (high risk group) and 1,114 samples from general people (low risk group) were examined for specific IgG antibodies against T. gondii by ELISA. The results revealed that the overall seroprevalence of T. gondii infection was 7.4% (n=1,787). The seroprevalence among low risk group was 8.0% (89/1,114), whereas that among high risk group was rather lower 6.4% (43/673), though this difference was statistically not significant (P=0.211). Among the risk factors, only the outdoor activity of pet cats was important; people having cats with outdoor activities revealed 2 times higher seroprevalence than people having cats with only indoor activities (P=0.027). In conclusion, the seroprevalence of T. gondii was not significantly different between the high risk group and low risk group, and the importance of cats as a source of infection in Korea is questionable.

Prevalence of Intestinal Protozoans among Schoolchildren in Suburban Areas near Yangon, Myanmar.

Although intestinal protozoans are common etiologies of diarrhea, few studies have been conducted in Myanmar. This study planned to investigate the prevalence of Giardia lamblia, Entamoeba coli, Entamoeba histolytica, and Endolimax nana among schoolchildren and their guardians in suburban areas near Yangon, Myanmar. We performed a cross-sectional survey among schoolchildren and their guardians from 7 primary schools in South Dagon and Hlaing Thar Yar districts, Yangon, Myanmar. Stool samples were observed with a microscope after concentration technique and iodine staining. Total 821 stool samples, including 556 from schoolchildren and 265 from guardians, were examined. The median age was 6 years old for schoolchildren and 36 years old for guardians. A 53.1% of the school children and 14.6 % of the guardians were males. The overall prevalence of each intestinal protozoan species was as follows: 3.4% (28/821) for G. lamblia; 3.5% (29/821) for E. coli; 1.2% (10/821) for E. histoytica, and 3.0% for E. nana. This study showed that intestinal protozoans are common in primary schoolchildren and their guardians in suburban areas near Yangon, Myanmar. Health interventions, such as hand washing education, improvement of sanitation, and establishment of water purification systems are urgently needed in this area.

Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes.

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.

High Toxoplasma gondii Seropositivity among Brain Tumor Patients in Korea.

Toxoplasma gondii is an intracellular protozoan that can modulate the environment of the infected host. An unfavorable environment modulated by T. gondii in the brain includes tumor microenvironment. Literature has suggested that T. gondii infection is associated with development of brain tumors. However, in Korea, epidemiological data regarding this correlation have been scarce. In this study, in order to investigate the relationship between T. gondii infection and brain tumor development, we investigated the seroprevalence of T. gondii among 93 confirmed brain tumor patients (various histological types, including meningioma and astrocytoma) in Korea using ELISA. The results revealed that T. gondii seropositivity among brain tumor patients (18.3%) was significantly (P<0.05) higher compared with that of healthy controls (8.6%). The seropositivity of brain tumor patients showed a significant age-tendency, i.e., higher in younger age group, compared with age-matched healthy controls (P<0.05). In conclusion, this study supports the close relationship between T. gondii infection and incidence of brain tumors.

Toxoplasma gondii B1 Gene Detection in Feces of Stray Cats around Seoul, Korea and Genotype Analysis of Two Laboratory-Passaged Isolates.

The increasing prevalence of Toxoplasma gondii infection in the human population in the Republic of Korea (= Korea) is due to various reasons such as an increase in meat consumption. However, the importance of cats in transmitting T. gondii infection through oocysts to humans has seldom been assessed. A total of 300 fecal samples of stray cats captured around Seoul from June to August 2013 were examined for T. gondii B1 gene (indicating the presence of oocysts) using nested-PCR. Fourteen (4.7%) of 300 cats examined were positive for B1 gene. Female cats (7.5%) showed a higher prevalence than male cats (1.4%). Cats younger than 3 months (5.5%) showed a higher prevalence than cats (1.5%) older than 3 months. For laboratory passage of the positive samples, the fecal suspension (0.2 ml) of B1 gene positive cats was orally inoculated into experimental mice. Brain tissues of the mice were obtained after 40 days and examined for the presence of tissue cysts. Two isolates were successfully passaged (designated KNIH-1 and KNIH-2) and were molecularly analyzed using the SAG5D and SAG5E gene sequences. The SAG5D and SAG5E gene sequences showed high homologies with the ME49 strain (less virulent strain). The results indicated the importance of stray cats in transmitting T. gondii to humans in Korea, as revealed by detection of B1 gene in fecal samples. T. gondii isolates from cats were successfully passaged in the laboratory for the first time in Korea.